Herpes simplex viruses type 1 and 2 (HSV-1 and HSV-2) are human pathogens that establish lifelong latent infections and cause a number of ailments. Infection is initiated by the merger of the viral envelope and a host cell membrane, which is catalyzed by the viral fusogen gB with the help of additional proteins, gD and gH/gL plus a cellular receptor for gD. By analogy with other viral fusogens, gB is thought to refold from the prefusion to the postfusion form in a series of large conformational changes that energetically couple refolding to membrane fusion. While the structure of the postfusion form is known, the prefusion form has not yet been characterized. The lack of the prefusion structure of gB is a major gap in our knowledge that has hindered the deciphering of the herpesvirus cell entry mechanism. The long-term goal of this research is the elucidation of the atomic-level mechanism of membrane fusion during herpesvirus entry and cell spread. The objective of this proposal is to stabilize the prefusion form of HSV-1 gB and to obtain its structure. Approaches that worked for other viral fusogens have so far failed with gB; thus, new strategies towards obtaining the prefusion structure of gB are needed. This R21 proposal is driven by an innovative central hypothesis that the prefusion form of gB is stabilized by interaction with membrane and requires the presence of membrane-proximal, transmembrane, and cytoplasmic domains. This hypothesis will be examined in two Specific Aims: Aim 1 will use gB-pseudotyped VSV?G virions, a novel reagent generated in preliminary data, to obtain a low-resolution cryoET reconstruction of the prefusion form of gB in the viral envelope to determine its overall architecture of the prefusion gB in its native-like state. In Aim 2, membrane mimetics - detergent/lipid micelles and nanodiscs - will be used to extract the soluble, prefusion form of the full-length gB expressed in insect cells. This form will be used in crystallization trials and in cryoEM single-particle 3D reconstruction, which will be used to generate a pseudoatomic model. In addition to the structure of the prefusion gB ectodomain, the proposed work will also yield the structures of the membrane-proximal, transmembrane, and cytoplasmic domains. This proposal is innovative because of its novel hypothesis and innovative approaches aimed at stabilizing the prefusion form. This proposal is significant because it addresses an important problem that has impeded the unraveling of the cell entry mechanism of HSV and other herpesviruses. The structure of the prefusion form of gB, at any resolution, would be an important discovery with a major impact on the herpesvirus field. It would inform the future work of many other investigators by providing a 3D framework for mapping functional data. Moreover, it may finally explain why gB requires other proteins to function as a fusogen. The results obtained here will form the basis for a future R01 proposal to determine the atomic-level crystal structure of prefusion gB and to elucidate its fusogenic mechanism using structural and biophysical approaches. Beyond the structure, finding a strategy to stabilize gB in its prefusion form may pave the way to a successful subunit vaccine against HSV.